Tyrosine kinases are regulatory proteins that play a central role in cell growth and differentiation. In preliminary work, the expression of tyrosine kinases in breast cancer cell lines was compared to normal-like breast cells using a tyrosine kinase differential display assay. The nuclear tyrosine kinase Rak was downregulated or not expressed in several breast cancer cell lines. Furthermore, the adenovirus early gene E1A, which is known to inhibit the growth of breast cancer cell lines, upregulated Rak expression. Enhanced expression of Rak in breast cancer cells had a growth suppressing activity as measured by a decrease in colony formation efficiency. Surprisingly, little is known about the role of Rak in cells and its mechanism of action. This proposal will test the hypothesis that Rak inhibits tumor cell growth, and that loss of expression of Rak plays a role in breast cancer. First, the effect of Rak expression on tumor cell growth will be assessed in vitro by cell counts, the colony formation assay and the soft agar assay, and tested in vivo. Second, the effect of Rak on the sensitivity of breast cancer cells to apoptosis and on the cell cycle will be studied. The contribution of critical domains of Rak to its function will be studied by deletion and mutation analysis. Third, expression of Rak in breast cancers of various grades and metastatic potentials will be determined by immunohistochemical analysis. These studies will delineate the role of Rak in cell growth and its mechanism of action. From a scientific standpoint, determining Rak's mechanism of action will significantly enhance our understanding of how cell growth is regulated. From a clinical standpoint, Rak may be a useful prognostic marker, and a gene therapy agent. Furthermore, identifying Rak's role in tumor growth will allow us to better tailor the specificity of tyrosine kinase inhibitors used in clinical trials.